The medaka fish (Oryzias latipes) is a teleost inhabiting in East Asia and has been used for studying vertebrate genetics. The mutations at the i locus of the medaka fish cause amelanotic skin and red-colored eyes. This i locus is known to encode a gene for tyrosinase. From one of the i alleles, i4, DNA of about 4.7-kb was cloned and found to have a transposon-like sequence; that is to say, it included open reading frames homologous to transposases of transposons of the hAT family including hobo of Drosophila, Ac of maize and Tam3 of snapdragon, and short terminal inverted repeats. This medaka element was named Tol2. The laboratory strains of the medaka fish contain about 10 copies of this element per haploid genome.
In the i4 mutant fish, the Tol2 element found in the tyrosinase gene locus has been shown by PCR to be excised from the target locus during embryonic development (Koga et al., 1996).
Zebrafish (Danio rerio), as well as the medaka fish (Oryzias latipes), is a small teleost and has been developed as a model animal to study vertebrate genetics and development (Takeuchi, 1966; Yamamoto, 1967; Streisinger et al., 1981). In zebrafish, large-scale chemical mutagenesis screens have been performed (Driever et al., 1996; Haffter et al., 1996), and, to facilitate cloning of the mutated genes, an insertional mutagenesis method using a pseudotyped retrovirus has been developed and performed (Lin et al., 1994; Gaiano et al., 1996; Amsterdam et al., 1997). Also, in an attempt to develop transposon technologies that would allow enhancer trap and gene trap screens to be performed, transposition of transposons of the Tcl/mariner family in fish has been tested and demonstrated (Ivics et al., 1997; Raz et al., 1997; Fadool et al., 1998). Although these results are encouraging, neither highly efficient transgenesis nor insertional mutagenesis methods using a transposon have not yet been developed.
The present inventors have been interested in developing novel transposon technologies using the Tol2 element. As a first step towards this goal, the present inventors developed a transient embryonic excision assay using zebrafish embryos, in which zebrafish fertilized eggs were injected with a plasmid DNA harboring the Tol2 element, showed that the Tol2 element was excisable from the injected plasmid DNA, and indicated that the Tol2 element is an autonomous member and is active in zebrafish (Kawakami et al., (1998) Gene 225, 17-22). Although the DNA sequence of the Tol2 element is similar to those of transposases of transposons of the hAT family, neither an active enzyme, which can function in trans, nor cis-elements essential for the excision reaction have been identified. In order to develop the Tol2 element as a useful tool for transgenesis and insertional mutagenesis, it is necessary to dissect and characterize cis and trans requirements. The functional transposase encoded by the Tol2 element had not yet been identified prior to the present invention.